The present invention relates to a methodology for high fidelity cloning of target nucleic acids. The invention involves application of polymerase chain reaction (PCR) for a few cycles, which minimizes number of doublings of target sequences, and hence greatly reduces generation of polymerase-induced mutant fraction in PCR products. The invention also describes cloning of such high fidelity PCR products in a positive selection vector.
Polymerase chain reaction or PCR (Saiki et al., 1985, Science 230, 1350-1354; Mullis and Faloona, 1987, Method Enzymol. 155, 335-350; U.S. Pat. Nos. 4,683,195; 4,683,202 and 4,965,188) has revolutionized amplification of target nucleic acids. The technique involves repeated cycles of denaturation of template nucleic acid molecules, sequence-specific primer annealing and primer extension using DNA polymerase thus resulting in an exponential amplification of the target nucleic acids. Usually, 30 cycles of PCR result in one million-fold amplification of target sequences from 20 doublings.
The PCR product itself could be used for diagnosis, quantitation of the template, direct sequencing and some other applications (U.S. Pat. Nos. 5,856,144; 5,487,993 and 5,891,687). However, for applications such as mutation analysis, identification of polymorphic transcripts, making RNA probes, sequencing, gene expression etc., usually a large quantity of DNA is needed. Thus it is necessary to isolate a bacterial clone carrying the PCR generated target DNA fragment in a cloning vector.
For correct structural and functional analyses it is fundamentally important to clone wild-type nucleic acids. Cloning of wild-type nucleic acids is critical for mutation analysis, identification of polymorphic transcripts and sequencing. It is also of paramount importance that for accurate functional analysis a true copy of the desired gene should be cloned in a suitable expression vector. However, an inherent disadvantage of DNA amplification using PCR is the introduction of mutations by the polymerases during synthesis of new DNA (Kunkel and Bebenek, 2000, Annu. Rev. Biochem. 69, 497-529; Andre et al., 1997, Genome Res. 7, 843-852; Cline et al., 1996, Nucl. Acids Res. 24, 3546-3551; Kong et al., 1993, J. Biol. Chem. 268, 1965-1975; Cariello et al., 1991, Nucl. Acids Res. 19, 4193-4198; Lundberg et al., 1991, Gene 180, 1-6; Reiss et al., 1990, Nucl. Acids Res. 18, 973-978; Keohavong and Thilly, 1989, Proc. Natl. Acad. Sci. USA 86, 9253-9257; Keohavong et al., 1988, DNA 7, 63-70; Kunkel et al., 1984, J. Bio. Chem. 259, 1539-1545; Loeb and Kunkel, 1982, Annu. Rev. Biochem. 52, 429-457). The total mutant fraction in a newly synthesized PCR-amplified DNA pool depends on the length of target sequence, error rate of a DNA polymerase, and number of doublings of the target sequence (Kunkel and Bebenek, 2000, Annu. Rev. Biochem. 69, 497-529; Andre et al., 1997, Genome Res. 7, 843-852; Cariello et al., 1991, Nucl. Acids Res. 19, 4193-4198; Reiss et al., 1990, Nucl. Acids Res. 18, 973-978). The error rates of different DNA polymerases vary from 1.3xc3x9710xe2x88x924 to 6.5xc3x9710xe2x88x927 mutant per basepair per doubling (Andre et al., 1997, Genome Res. 7, 843-852; Cline et al., 1996, Nucl. Acids Res. 24, 3546-3551; Cariello et al., 1991, Nucl. Acids Res. 19, 4193-4198; Keohavong and Thilly, 1989, Proc. Natl. Acad. Sci. USA 86, 9253-9257). For a million-fold amplification of target sequences 20 doublings are required, which are usually achieved by 30 cycles of PCR amplification. For a specific target sequence the increase of mutation fraction in the PCR-amplified DNA pool is a linear function of the number of doublings of the target sequence provided that the error rate of DNA polymerase remains constant under the specific PCR conditions (Kunkel and Bebenek, 2000, Annu. Rev. Biochem. 69,497-529; Andre et al., 1997, Genome Res. 7, 843-852; Cariello et al., 1991, Nucl. Acids Res. 19, 4193-4198; Reiss et al., 1990, Nucl. Acids Res. 18, 973-978).
The present methods of PCR cloning involve usually cloning of PCR products obtained after 20 doublings of target sequences thus resulting in generation of significant number of mutant clones, especially in case of cloning large target DNA fragments. Consequently, sometime rigorous sequencing of many clones is required to isolate a correct clone, and very often site-directed mutagenesis is necessary to correct a mutant clone. Furthermore, failure of PCR cloning of a correct sequence necessitates laborious screening of genomic or cDNA libraries, which usually represent correct sequences.
The primary object of this invention is to develop a methodology for cloning of high fidelity PCR products that contain no or minimum mutations. The invention aims to generate high fidelity PCR products by amplifying target sequences only for a few cycles, which minimizes number of doublings of target sequences, and hence greatly reduces polymerase-induced mutant fraction in PCR products. The invention further aims to clone high fidelity PCR products in a suitable vector.
Elimination of disadvantages associated with present protocols of PCR cloning and library screening is greatly desirable. The present invention aims to achieve a milestone advancement in cloning of a target sequence with no mutation.
The present invention describes a methodology of high fidelity PCR cloning of target nucleic acids. During PCR amplification of target nucleic acid sequences the polymerase-induced mutation fraction is linearly proportional to the number of doublings of the target sequences. The invention uses PCR on target nucleic acid sequences only for a few cycles, which minimizes number of doublings of target sequences, and hence greatly reduces polymerase-induced mutant fraction in PCR products. The high fidelity PCR products thus obtained are then cloned into a suitable vector. As an example, a 960 bp target sequence from E. coli DNA was amplified using PCR only for 3 cycles, and it was then directly cloned into a positive selection cloning vector pRGR2Ap. All insert-carrying clones showed cloning of functionally wild-type target DNA sequences, which indicated that the cloned target sequences most probably contained no mutation. Cloning of PCR products obtained from 3 cycles of amplification, instead of 30 cycles of amplification, theoretically achieves 10-fold reduction of mutations in the cloned fragments. The invention also envisions cloning of high fidelity products of primer extension.
Not Applicable
The present invention is directed to develop a PCR cloning method that will greatly reduce, if not eliminate, the number of mutant clones that are observed with present PCR cloning experiments. Presently, in a typical PCR cloning experiment PCR products obtained usually from 30 cycles of PCR amplification are used in cloning. PCR amplification of target sequences for 30 cycles usually result in one million-fold amplification of target sequences, which is equivalent to 20 doublings of target sequences. The increase in total mutant fraction in PCR products pool is a linear function of the number of doublings of target sequences (Kunkel and Bebenek, 2000, Annu. Rev. Biochem. 69, 497-529; Andre et al., 1997, Genome Res. 7, 843-852; Cariello et al., 1991, Nucl. Acids Res. 19, 4193-4198; Reiss et al., 1990, Nucl. Acids Res. 18, 973-978). The invention describes generation of high fidelity PCR products by amplifying the target sequences only for a few cycles, which minimizes number of doublings of target sequences, and hence greatly reduces polymerase-induced mutant fraction in PCR products. The invention further describes cloning of such high fidelity PCR products in a suitable vector.
The amount of DNA obtained after a few cycles of PCR amplification is very small, and hence cloning of such small amount of DNA should give only a few colonies even at the most efficient conditions of ligation and transformation. Thus it was decided to clone such high fidelity PCR products into a positive selection cloning vector pRGR2Ap, which gives a very few false positive background colonies (Malo and Husain, 2000, USPTO application #09/722219). The positive selection vector pRGR2Ap has been developed based on reconstruction of the ampicillin resistance reporter gene. When the last (position 286) amino acid tryptophan (encoded by 5xe2x80x2-TGG-3xe2x80x2) of ampicillin resistance gene product xcex2-lactamase is replaced by valine (encoded by 5xe2x80x2-GTG-3xe2x80x2) xcex2-lactamase becomes functionally inactive. The sequence 5xe2x80x2-GTG-3xe2x80x2 is a part of the Pml I restriction endonuclease cleavage site 5xe2x80x2-CACGTG-3xe2x80x2, which is a unique cloning site in this vector. Thus upon Pml I restriction endonuclease cleavage 5xe2x80x2-CAC-3xe2x80x2 and 5xe2x80x2-GTG-3xe2x80x2 are created at the 3xe2x80x2 and 5xe2x80x2 ends respectively of the linearized blunt-ended vector. A PCR primer carrying the nucleotides 5xe2x80x2-TGGTAA-3xe2x80x2 at its 5xe2x80x2 end is used in PCR. When the resulting blunt-ended PCR products are ligated to the vector the reporter ampicillin resistance gene is reconstructed correcting the mutation. The nucleotides 5xe2x80x2-TAA-3xe2x80x2 constitute the stop codon for the ampicillin resistance gene. Subsequent transformation of a host cell with the recombinant vector (carrying an insert DNA) produces functionally active xcex2-lactamase, which confers resistance to ampicillin. Hence only the recombinant clones grow in an ampicillin-containing medium, and cloning in pRGR2Ap is unidirectional. Insertional reconstruction of the ampicillin resistance reporter gene in pRGR2Ap also minimizes generation of false positive clones arising from recircularization of linearized vectors digested by contaminating exonucleases present in restriction endonucleases, ligases, DNA polymerases and other reagents. In case of other available toxin-based positive selection vectors or lacZ-based chromogenic vectors, false positive clones arise from recircularization of linearized exonuclease-digested vectors, which could have lost some bases from their ends due to exonuclease digestion. Recircularized exonuclease-digested pRGR2Ap should not produce functionally active xcex2-lactamase, and hence should not grow in a medium containing ampicillin thus greatly reducing exonuclease-induced false positive clones. The vector pRGR2Ap carries the tetracycline resistance gene as the selectable marker gene.